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Background: Congenital cytomegalovirus (CMV) infection is the most prevalent congenital infection worldwide. Epidemiology and clinical outcomes are known to vary with socio-economic background, but few data are available from developing countries, where the overall burden of infectious diseases is frequently high. Methodology/Principal Findings: As part of an ongoing birth cohort study in The Gambia among term infants, urine samples were collected at birth and tested by PCR for the presence of CMV DNA. Risk factors for transmission and clinical outcome were assessed, including placental malaria infection. Babies were followed up at home monthly for morbidity and anthropometry, and at one year of age a clinical evaluation was performed. The prevalence of congenital CMV infection was 5.4% (40/741). A higher prevalence of hepatomegaly was the only significant clinical difference at birth. Congenitally infected children were more often first born babies (adjusted odds ratio (OR) 5.3, 95% confidence interval (CI) 2.0-13.7), more frequently born in crowded compounds (adjusted OR 2.9, 95%CI 1.0-8.3) and active placental malaria was more prevalent (adjusted OR 2.9, 95%CI 1.0-8.4). These associations were corrected for maternal age, bed net use and season of birth. During the first year of follow up, mothers of congenitally infected children reported more health complaints for their child. Conclusions/Significance: In this study, the prevalence of congenital CMV among healthy neonates was much higher than previously reported in industrialised countries, and was associated with active placental malaria infection. There were no obvious clinical implications during the first year of life. The effect of early life CMV on the developing infant in the Gambia could be mitigated by environmental factors, such as the high burden of other infections. © 2007 van der Sande et al.
In January 2002, a birth cohort was initiated in the village of Sukuta, The Gambia, approximately 15 km away from the Medical Research Council (MRC) Laboratories in Fajara, in order to study the epidemiological, clinical, immunological and virological determinants of early life CMV infection in an endemic environment. The wider objective of this cohort was to understand how the immune system of the foetus and new born develops in relation to CMV and other early life infections, and how this interacts with the response to vaccinations. Sukuta has approximately 25,000 inhabitants, and is adjacent to, but distinct from the nearby expanding peri-urban community centred around the village of Serrekunda between the capital Banjul 20 km to the north and a growing urban centre (Brikama) 25 km to the south. A variety of ethnic groups live in the village, although the Mandinka group make up about half of the population. Eligible for recruitment in the cohort were children born in the health centre of the study village whose parents gave informed consent. Babies whose mothers had suffered a serious infectious disease during pregnancy for which they had been admitted were not recruited. Pre-term babies, babies born with a serious congenital deficit, or babies who were in need for a transfer to a referral hospital immediately following delivery, were not included either. Routine follow up was conducted monthly. During these routine visits, anthropometric measurements and a morbidity checklist were completed. Vaccinations were given according to the Gambia Government schedule. Recruitment and follow-up are still ongoing. Urine was collected within two weeks of birth and transported to the laboratory within 24 hours, and stored at −20°C. CMV DNA was detected by an in-house nested PCR method amplifying a region of the UL50 gene. Frequent negative controls (one in seven reactions) were included to minimise the risk of false-positive reactions. CMV viral load in maternal samples collected at the time of delivery (vagina, urine, saliva, colostrum and plasma) from a sub-set of mothers was quantified by real-time PCR. The lower limit of detection by real-time PCR was 100 copies/ml for plasma and vaginal swab, and 50 copies/ml for the other samples. Maternal plasma levels of CMV-specific IgG were detected and quantified using commercial enzyme immunoassays (DiaSorin, Saluggia, Italy) used according to the manufacturer’s instructions. The complete results from maternal testing will be described in a subsequent viroimmunology paper. Immediately upon delivery, a placental imprint was made on a glass slide. Slides were Giemsa stained, and transported to the laboratory for the detection of parasitaemia. Slides were read by a trained microscopist for the speciation and quantification of malaria parasites; and a random selection of slides was read by a second trained microscopist to assure quality control. At least 100 fields were read before a negative result was declared. A placental biopsy was taken and immediately placed into 10% formalin for transport to the laboratory. Each placental sample was embedded in paraffin wax, sectioned and stained with standard H&E stain. Slides were examined under a light microscope for evidence of malaria infection according to the classification described by Ismail et al. [20]. Thus placentas were ascribed to one of 4 groups: no infection, acute infection (parasites in the intervillous space), chronic infection (parasites and malaria pigment) or past infection (pigment only). A paediatric assessment, including a neurological examination of the child was performed at birth by a qualified paediatrician (first OO, later MP), at which time the CMV status of the baby was not yet known. Baseline data were collected at birth, and every month, morbidity data and anthropometry was collected in a standardised way. Parents could consult the paediatrician with any complaints or concerns about their children’s health and welfare throughout the follow up period, and at one year of age another standardised clinical evaluation was performed by the paediatrician. Maternal height and weight were measured six months after delivery. Congenital CMV was defined as the detection of CMV in the urine by PCR within two weeks of birth. Acute malaria infection was defined as the detection of malaria parasites by microscopy, or the detection of an acute infection by histology. Active malaria infection was defined as a histological diagnosis of acute or chronic infection. Field, clinic and laboratory data were all merged and validated in a relational Microsoft Access database. Data were analysed using Stata 8 (Stata Corp, Texas, USA). Statistical significance was assigned when a p-value<0.05 was obtained. Differences in proportions were compared with a chi-squared test. Univariate logistic regression analysis was used to calculate odds ratio's (ORs) to test for significance of associations between risk factors and congenital CMV infection. Where needed, variables were dichotomised or categorised to enable inclusion in a logistic regression analysis. Risk factors were included in a stepwise backward multivariate model if p<0.1 to obtain independent adjusted ORs. The study was approved by the Gambia Government/MRC Ethics Committee. All parents gave written informed consent for their child to participate.
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